Tunable Substrate Functionalities Direct Stem Cell Fate toward Electrophysiologically Distinguishable Neuron-like and Glial-like Cells

Engineering cellular microenvironment on a functional platform using various biophysical cues to modulate stem cell fate has been the central theme in regenerative engineering. Among the various biophysical cues to direct stem cell differentiation, the critical role of physiologically relevant electric field (EF) stimulation was established in the recent past. The present study is the first to report the strategy to switch EF-mediated differentiation of human mesenchymal stem cells (hMSCs) between neuronal and glial pathways, using tailored functional properties of the biomaterial substrate. We have examined the combinatorial effect of substrate functionalities (conductivity, electroactivity, and topography) on the EF-mediated stem cell differentiation on polyvinylidene-difluoride (PVDF) nanocomposites in vitro, without any biochemical inducers. The functionalities of PVDF have been tailored using conducting nanofiller (multiwall-carbon nanotube, MWNT) and piezoceramic (BaTiO3, BT) by an optimized processing approach (melt mixing-compression molding-rolling). The DC conductivity of PVDF nanocomposites was tuned from ∼10-11 to ∼10-4 S/cm and the dielectric constant from ∼10 to ∼300. The phenotypical changes and genotypical expression of hMSCs revealed the signatures of early differentiation toward neuronal pathway on rolled-PVDF/MWNT and late differentiation toward glial lineage on rolled-PVDF/BT/MWNT. Moreover, we were able to distinguish the physiological properties of differentiated neuron-like and glial-like cells using membrane depolarization and mechanical stimulation. The excitability of the EF-stimulated hMSCs was also determined using whole-cell patch-clamp recordings. Mechanistically, the roles of intracellular reactive oxygen species (ROS), Ca2+ oscillations, and synaptic and gap junction proteins in directing the cellular fate have been established. Therefore, the present work critically unveils complex yet synergistic interaction of substrate functional properties to direct EF-mediated differentiation toward neuron-like and glial-like cells, with distinguishable electrophysiological responses.